The overall objective of this proposal is to systematically develop our understanding of gene regulation in virus-infected cells and to use this knowledge for developing in vitro systems from which activities can be purified and characterized. As this is a renewal of research in progress, all the outlined projects have already produced promising results. The following is a list of these projects which deal with four viruses: the papova viruses SV40 and polyoma, the parvovirus MVM and the retrovirus MLV. Studies with SV40 and polyoma: 1) Purification, characterization and biological functions of poly (A)-minus viral-specific RNAs. 2) Purification, characterization and biological functions of small viral RNAs. 3) Purification, characterization and biological function of high molecular weight SV40 RNA. 4) "Attenuation" as a mechanism for regulating transcription. 5) Isolation, purification and characterization of various forms of viral minichromosomes. 6) Characterization of minichromosomes of substituted SV40 DNA molecules. 7) Characterization of minichromosomes having transcriptional activities. 8) Transcriptional initiation in vitro using either E. coli polymerase or eukaryotic polymerase II and viral minichromosomes. 9) The relation of the cytoskeleton structure to mRNA biogenesis and cellular architectural features that affect genomic activity. 10) Analysis of polyoma nuclear RNA. Studies with MVM: 1) Characterization of early and late viral RNAs. 2) Transcriptional inition and capping of MVM RNAs. 3) Isolation and characterization of transcriptional complexes. 4) Splicing as a mechanism regulating permisivity. 5) Isolation, characterization and biological functions of "lollipop" and circular MVM DNA structures. 6) The possibility that MVM DNA has a minichromosome structure. Studies with MLV: Characterization of poly (A)-ion and poly (A)-minus viral RNAs. In addition, the viral RNAs of the various systems will be used as substrates in in vitro studies aimed at isolation and characterization of splicing activity. The various RNAs that will be used are: a) nuclear and cytoplasmic 19S SV40 RNAs; b) Nuclear MVM RNA; and c) MLV genomic 35S RNA.